U.S. patent application number 12/367867 was filed with the patent office on 2009-11-12 for cysteine and cystine prodrugs to treat schizophrenia and reduce drug cravings.
Invention is credited to David A. Baker, James M. Cook, Edward Merle Johnson, II, Wenyuan Yin.
Application Number | 20090281109 12/367867 |
Document ID | / |
Family ID | 40661413 |
Filed Date | 2009-11-12 |
United States Patent
Application |
20090281109 |
Kind Code |
A1 |
Cook; James M. ; et
al. |
November 12, 2009 |
CYSTEINE AND CYSTINE PRODRUGS TO TREAT SCHIZOPHRENIA AND REDUCE
DRUG CRAVINGS
Abstract
The present invention provides cysteine and cystine prodrugs for
the treatment of schizophrenia and drug addiction. The invention
further encompasses pharmaceutical compositions containing prodrugs
and methods of using the prodrugs and compositions for treatment of
schizophrenia and drug addiction.
Inventors: |
Cook; James M.; (Milwaukee,
WI) ; Baker; David A.; (Grafton, WI) ; Yin;
Wenyuan; (Milwaukee, WI) ; Johnson, II; Edward
Merle; (Glendale, WI) |
Correspondence
Address: |
QUARLES & BRADY LLP
411 E. WISCONSIN AVENUE, SUITE 2040
MILWAUKEE
WI
53202-4497
US
|
Family ID: |
40661413 |
Appl. No.: |
12/367867 |
Filed: |
February 9, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61026874 |
Feb 7, 2008 |
|
|
|
Current U.S.
Class: |
514/252.11 ;
514/252.1; 514/547; 514/550; 544/349; 544/357; 544/385; 544/408;
560/147 |
Current CPC
Class: |
C07D 241/08 20130101;
A61P 25/30 20180101; C07K 5/0606 20130101; A61P 25/18 20180101;
C07D 487/04 20130101; C07C 323/60 20130101; C07D 211/86 20130101;
A61P 43/00 20180101; A61K 9/0053 20130101; C07C 327/34
20130101 |
Class at
Publication: |
514/252.11 ;
544/408; 544/349; 544/385; 544/357; 514/252.1; 560/147; 514/550;
514/547 |
International
Class: |
A61K 31/497 20060101
A61K031/497; C07D 241/08 20060101 C07D241/08; C07D 241/36 20060101
C07D241/36; C07D 241/04 20060101 C07D241/04; A61K 31/4965 20060101
A61K031/4965; C07C 69/003 20060101 C07C069/003; A61K 31/215
20060101 A61K031/215; A61K 31/225 20060101 A61K031/225; A61P 25/18
20060101 A61P025/18; A61P 25/30 20060101 A61P025/30 |
Claims
1. A cysteine prodrug having the structure: ##STR00046## wherein:
R.sup.1 and R.sup.2 are independently selected from OH, .dbd.O, or
a branched or straight chain C.sub.1 to C.sub.5 alkoxyl group, with
the caveat that when .dbd.O is selected the nitrogen atom adjacent
the carbonyl group thusly formed bears a H and a single bond joins
the adjacent nitrogen to said carbonyl group; R.sup.3 is H, a
branched or straight chain C.sub.1 to C.sub.5 alkyl, a
nitrobenzenesulfonyl, a trityl, an aryl thio, an aryl, an
alkylthio, an acyl, a benzoyl, a thio acyl, a thio benzoyl, or a
benzyl group; and R.sup.4 is selected from the side chain groups of
the natural L-amino acids cys, gly, phe, pro, val, ser, arg, asp,
asn, glu, gln, ala, his, ile, leu, lys, met, thr, trp, tyr, or
D-isomers thereof, with the caveat that when R.sup.4 is the side
chain group of the natural L-amino acid gly, R.sup.1 and R.sup.2
are not both selected to be .dbd.O; or a cystine dimer of said
prodrug having the structure: ##STR00047## wherein: R.sup.1,
R.sup.2, R.sup.5 and R.sup.6 are independently selected from OH,
.dbd.O, or a branched or straight chain C.sub.1 to C.sub.5 alkoxyl
group, with the caveat that when .dbd.O is selected the nitrogen
atom adjacent the carbonyl group thusly formed bears a H and a
single bond joins the adjacent nitrogen to said carbonyl group; and
R.sup.4 and R.sup.7 are independently selected from the side chain
groups of the natural L-amino acids cys, gly, phe, pro, val, ser,
arg, asp, asn, glu, gln, ala, his, ile, leu, lys, met, thr, trp,
tyr, or D-isomers thereof, with the caveat that when R.sup.4 and
R.sup.7 are both the side chain group of the natural L-amino acid
gly, R.sup.1, R.sup.2, R.sup.5 and R.sup.6 shall not all be
selected be .dbd.O.
2. The cysteine prodrug according to claim 1, wherein said cysteine
prodrug has the structure: ##STR00048##
3. The cysteine prodrug according to claim 1, wherein said cysteine
prodrug is in the form of the cystine dimer having the structure:
##STR00049##
4. The cysteine prodrug according to claim 1, wherein said cysteine
prodrug in the form of the cystine dimer includes identical R.sup.4
and R.sup.7 groups.
5. The cysteine prodrug according to claim 1, wherein said cysteine
prodrug in the form of the cystine dimer includes non-identical
R.sup.4 and R.sup.7 groups.
6. The cysteine prodrug according to claim 1, wherein said cysteine
prodrug or cystine dimer thereof includes at least one R.sup.4 and
R.sup.7 group that is a cys, said cys further protected by a
branched or straight chain C.sub.1 to C.sub.5 alkyl, a
nitrobenzenesulfonyl, a trityl, an aryl thio, an aryl, an
alkylthio, an acyl, a benzoyl, a thio acyl, a thio benzoyl, or a
benzyl group.
7. A method of reducing schizophrenia in a subject comprising
administering to said subject an effective amount of a cysteine
prodrug or cystine dimer thereof according to claim 1, whereby
schizophrenia is reduced in said subject.
8. The method according to claim 7, wherein the step of
administering to said subject is accomplished by oral delivery.
9. A pharmaceutical composition comprising a cysteine prodrug or
cystine dimer thereof according to claim 1 and a
pharmaceutically-acceptable carrier.
10. A method of reducing drug craving in a subject comprising
administering to said subject an effective amount of a cysteine
prodrug or cystine dimer thereof according to claim 1, whereby drug
craving is reduced in said subject.
11. The method according to claim 10, wherein the step of
administering to said subject is accomplished by oral delivery.
12. A protected cysteine analog having the structure: ##STR00050##
or a cystine dimer of said protected cysteine analog having the
structure: ##STR00051## wherein R.sup.1 through R.sup.6 are
independently selected from a branched or straight chain C.sub.1 to
C.sub.5 alkyl, a phenyl, or a benzyl group.
13. The protected cysteine analog according to claim 12, wherein
said protected cysteine analog has the structure: ##STR00052##
14. The protected cysteine analog according to claim 12, wherein
said protected cysteine analog is in the form of the cystine dimer
having the structure: ##STR00053##
15. A method of reducing schizophrenia in a subject comprising
administering to said subject an effective amount of a protected
cysteine analog or cystine dimer thereof according to claim 12,
whereby schizophrenia is reduced in said subject.
16. The method according to claim 15, wherein the step of
administering to said subject is accomplished by oral delivery.
17. A pharmaceutical composition comprising a protected cysteine
analog or cystine dimer thereof according to claim 12 and a
pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional application 61/026,874, filed Feb. 7, 2008, which is
incorporated herein by reference in its entirety for all
purposes.
FIELD OF THE INVENTION
[0002] This invention relates generally to the treatment of
schizophrenia and drug addiction. More particularly, the present
invention is directed to cysteine and cystine prodrugs useful as
antipsychotic medications in the treatment of schizophrenia. As
well, the respective prodrugs are applicable for reducing drug
cravings in drug addicted individuals.
BACKGROUND OF THE INVENTION
[0003] Schizophrenia is a debilitating disorder afflicting 1% of
the world's population. The development of effective medications to
treat schizophrenia is reliant on advances in characterizing the
underlying pathophysiology. Chlorpromazine and other phenothiazines
are considered first generation antipsychotics (termed "typical
antipsychotics") useful in the treatment of schizophrenia. However,
the antipsychotic efficacy of phenothiazines was, in fact,
serendipitously discovered. These drugs were initially used for
their antihistaminergic properties and later for their potential
anesthetic effects during surgery. Hamon and colleagues extended
the use of phenothiazines to psychiatric patients and quickly
uncovered the antipsychotic properties of these compounds; shortly
thereafter, the pharmacologic characteristic of dopamine receptor
blockade was linked to the antipsychotic action of chlorpromazine
(Thorazine). This led to the development of additional dopamine
receptor antagonists, including haloperidol (Haldol). For nearly
fifty years, dopamine antagonists were the standard treatment for
schizophrenia even though these drugs induce severe side effects
ranging from Parkinson's disease-like motor impairments to sexual
dysfunction and are only effective in treating the positive
symptoms of schizophrenia.
[0004] In the 1970's, clozapine became the first "atypical
psychotic" or 2nd generation antipsychotic agent introduced.
Clinical trials have shown that clozapine produces fewer motor side
effects and exhibits improved efficacy against positive and
negative symptoms relative to 1st generation compounds. However,
clozapine was briefly withdrawn from the market because of the
potential to produce severe agranulocytosis, a potentially fatal
side effect requiring patients to undergo routine, costly
hematological monitoring. As a result, clozapine is only approved
for treatment-resistant schizophrenia. Although also a dopamine
receptor antagonist, the therapeutic site of action for clozapine
is thought to involve blockade of serotonin receptors. This led to
the generation of other serotonin receptor antagonists in the
1990's with the goal of improving the safety profile of
clozapine.
[0005] The growth potential for novel antipsychotics was revealed
following the introduction of risperidone in 1994; within two years
risperidone overtook haloperidol in the number of prescriptions
written by physicians. While it was generally assumed that the
newer 2nd generation antipsychotics also exhibited the favorable
efficacy profile produced by clozapine, the clinical data was
ambiguous. As a result, the NIH recently funded a large, lengthy,
and expensive clinical trial to examine this assumption. The
results of the Clinical Antipsychotic Trials of Intervention
Effectiveness (CATIE), recently released, indicate that there is no
benefit to the newer 2nd generation compounds. Specifically, 1st
and 2nd generation drugs did not differ in the incidence of severe
motor side-effects nor were 2nd generation agents found to be more
effective than 1st generation antipsychotics. In the CATIE trial,
74% of the patients discontinued treatment prior to completing the
18 month trial, in part due to a lack of efficacy and
intolerability of the treatment regimen.
[0006] As can be appreciated from the foregoing, there exists a
pressing need and considerable market potential for novel
antipsychotic agents. Of course, the development of effective
antipsychotic agents will be facilitated by a thorough
understanding of pathophysiologies underlying the neurological
disorders.
SUMMARY OF THE INVENTION
[0007] The present invention is based on the inventors' success in
identifying prodrugs of cysteine and cystine with utility as
antipsychotic and addiction reducing agents. Accordingly, the
invention provides a cysteine prodrug having the structure:
##STR00001##
wherein: R.sup.1 and R.sup.2 are independently selected from OH,
.dbd.O, or a branched or straight chain C.sub.1 to C.sub.5 alkoxyl
group, with the caveat that when .dbd.O is selected the nitrogen
atom adjacent the carbonyl group thusly formed bears a H and a
single bond joins the adjacent nitrogen to said carbonyl group;
R.sup.3 is H, a branched or straight chain C.sub.1 to C.sub.5
alkyl, a nitrobenzenesulfonyl, a trityl, an aryl thio, an aryl, an
alkylthio, an acyl, a benzoyl, a thio acyl, a thio benzoyl, or a
benzyl group; and R.sup.4 is selected from the side chain groups of
the natural L-amino acids cys, gly, phe, pro, val, ser, arg, asp,
asn, glu, gln, ala, his, ile, leu, lys, met, thr, trp, tyr, or
D-isomers thereof, with the caveat that when R.sup.4 is the side
chain group of the natural L-amino acid gly, R.sup.1 and R.sup.2
are not both selected to be .dbd.O; or a cystine dimer of said
prodrug having the structure:
##STR00002##
wherein: R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are independently
selected from OH, .dbd.O, or a branched or straight chain C.sub.1
to C.sub.5 alkoxyl group, with the caveat that when .dbd.O is
selected the nitrogen atom adjacent the carbonyl group thusly
formed bears a H and a single bond joins the adjacent nitrogen to
said carbonyl group; and R.sup.4 and R.sup.7 are independently
selected from the side chain groups of the natural L-amino acids
cys, gly, phe, pro, val, ser, arg, asp, asn, glu, gln, ala, his,
ile, leu, lys, met, thr, trp, tyr, or D-isomers thereof, with the
caveat that when R.sup.4 and R.sup.7 are both the side chain group
of the natural L-amino acid gly, R.sup.1, R.sup.2, R.sup.5 and
R.sup.6 shall not all be selected to be .dbd.O.
[0008] In certain preferred embodiments, the cysteine prodrug
according to the invention has the structure:
##STR00003##
[0009] The cysteine prodrug may alternatively be provided in the
form of a cystine dimer. Certain preferred cystine dimers according
to the invention have the structure: the form of the cystine dimer
having the structure:
##STR00004##
[0010] The invention provides synthetic routes for the synthesis of
cystine dimers having identical R.sup.4 and R.sup.7 groups or,
alternatively, mixed or non-identical R.sup.4 and R.sup.7
groups.
[0011] In certain cysteine prodrugs or cystine dimers of the
invention, at least one R.sup.4 and R.sup.7 group is a cys and the
reactive moiety is further protected by a branched or straight
chain C.sub.1 to C.sub.5 alkyl, a nitrobenzenesulfonyl, a trityl,
an aryl thio, an aryl, an alkylthio, an acyl, a benzoyl, a thio
acyl, a thio benzoyl, or a benzyl group.
[0012] In another aspect, the present invention provides a method
of reducing schizophrenia in a subject. Such a method includes
steps of administering to the subject an effective amount of a
cysteine prodrug or cystine dimer thereof according to the
invention, whereby schizophrenia is reduced in the subject.
Administration is preferably accomplished by oral delivery.
[0013] In yet another aspect, the invention provides a method of
reducing drug craving in a subject. Such a method includes steps of
administering to the subject an effective amount of a cysteine
prodrug or cystine dimer of the invention, whereby drug craving is
reduced in the subject. Again, administration is preferably via the
oral route.
[0014] Of course, the present invention encompasses pharmaceutical
compositions including a cysteine prodrug or cystine dimer
according to the invention in combination with at least a
pharmaceutically-acceptable carrier. The invention further
contemplates methods for the manufacture of such a pharmaceutical
composition for the reduction of schizophrenia and/or drug craving
in a subject.
[0015] A further aspect of the invention encompasses protected
cysteine analogs having the structure:
##STR00005##
or a cystine dimer of the protected cysteine analog having the
structure:
##STR00006##
[0016] wherein R.sup.1 through R.sup.6 are independently selected
from a branched or straight chain C.sub.1 to C.sub.5 alkyl, a
phenyl, or a benzyl group.
[0017] Preferable protected cysteine analogs according to the
invention have the structure:
##STR00007##
[0018] Alternatively, protected cysteine analogs may be provided in
the form of the corresponding cystine dimers. Certain preferred
cystine dimers have the structures:
##STR00008##
[0019] Related to the protected cysteine analogs, the invention
further provides a method of reducing schizophrenia in a subject by
administering to a subject an effective amount of a protected
cysteine analog or cystine dimer thereof according to the
invention, whereby schizophrenia is reduced in said subject.
Administration is preferably via the oral route.
[0020] The invention is also directed to protected cysteine analogs
or cystine dimers thereof having any one of the structures
described and claimed herein. Such analogs are useful in methods of
reducing schizophrenia or reducing drug cravings in a subject
comprising administering to the subject an effective amount of the
protected cysteine analog or cystine dimer.
[0021] The invention further encompasses pharmaceutical
compositions containing a protected analog or dimer thereof in
combination with a pharmaceutically-acceptable carrier. Methods of
formulating/manufacturing such pharmaceutical compositions for the
treatment of schizophrenia or for reducing drug craving in a
subject are also within the invention's scope.
[0022] Other objects, features and advantages of the present
invention will become apparent after review of the specification,
claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 depicts diketopiperazine targets based on various
natural L-amino acids and D-isomers thereof.
[0024] FIG. 2 illustrates general formulas for monomer and dimer
prodrugs and precursors thereof.
[0025] FIG. 3 provides general formulas for various protected
cysteine analogs.
[0026] FIG. 4 illustrates percent inhibition of a startle response
elicited by a loud auditory stimulus (50 dB above background) when
preceded by a mild auditory stimulus (2-15 dB above background) in
rats treated with pcp (0-2.0 mg/kg, N=9-60/group). * from every
other group at respective prepulse intensity, Fisher LSD
p<0.05
[0027] FIG. 5 displays the impact of N-acetyl cysteine on
sensorimotor gating deficits produced by phencyclidine administered
orally (left) or directly into the prefrontal cortex (right), which
is likely the therapeutic site of action for cysteine prodrugs. *
from every pcp only group at respective prepulse intensity, Fisher
LSD p<0.05
[0028] FIG. 6 illustrates the efficacy of exemplary compounds from
Scheme 1 relative to N-acetyl cysteine in reversing PCP-induced
deficits in sensorimotor gating in rats. * from every pcp only
group at respective prepulse intensity, +NAC 30 group, Fisher LSD
p<0.05
[0029] FIG. 7 shows the efficacy of exemplary compounds from Scheme
2 relative to N-acetyl cysteine in reversing PCP-induced deficits
in sensorimotor gating in rats. * from every pcp only group at
respective prepulse intensity, +NAC 30 group, Fisher LSD
p<0.05
[0030] FIG. 8 illustrates the efficacy of exemplary compounds from
Scheme 3 relative to N-acetyl cysteine in reversing PCP-induced
deficits in sensorimotor gating in rats. * from every pcp only
group at respective prepulse intensity, +NAC 30 group, Fisher LSD
p<0.05
[0031] FIG. 9 shows the efficacy of exemplary compounds from Scheme
4 relative to N-acetyl cysteine in reversing PCP-induced deficits
in sensorimotor gating in rats. * from every pcp only group at
respective prepulse intensity, +NAC 30 group, Fisher LSD
p<0.05
[0032] FIG. 10 illustrates the efficacy of compound from scheme 5
relative to N-acetyl cysteine in reversing PCP-induced deficits in
sensorimotor gating in rats. * from every pcp only group at
respective prepulse intensity, +NAC 30 group, Fisher LSD p<0.05
FIG. 11 provides a bar graph illustrating that N-acetylcysteine
(IP) is effective in producing a significant reduction in
cocaine-induced reinstatement at the doses of 30 and 60 mg/kg.
[0033] FIG. 12 depicts a bar graph illustrating that
N-acetylcysteine is less effective when given orally. Further,
administration of 1 mg/kg of Compound 5a-D (Scheme 1) was
sufficient to block cocaine-induced reinstatement, an effect that
was comparable to 30 mg/kg NAC.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Before the present materials and methods are described, it
is understood that this invention is not limited to the particular
methodology, protocols, materials, and reagents described, as these
may vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims.
[0035] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. As well,
the terms "a" (or "an"), "one or more" and "at least one" can be
used interchangeably herein. It is also to be noted that the terms
"comprising", "including", and "having" can be used
interchangeably.
[0036] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications and patents specifically mentioned herein are
incorporated by reference for all purposes including describing and
disclosing the chemicals, cell lines, vectors, animals,
instruments, statistical analysis and methodologies which are
reported in the publications which might be used in connection with
the invention. All references cited in this specification are to be
taken as indicative of the level of skill in the art. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0037] The term "lower alkyl group(s)" as used herein indicates a
linear, branched or cyclic alkyl group(s) having 1 to 6 carbon
atoms. They include, for example, methyl group, ethyl group,
n-propyl group, n-butyl group, n-pentyl group, n-hexyl group,
isopropyl group, isobutyl group, sec-butyl group, tert-butyl group,
isopentyl group, tert-pentyl group, neopentyl group, 2-pentyl
group, 3-pentyl group, 3-hexyl group, 2-hexyl group, cyclopropyl
group, cyclobutyl group, cyclopentyl group and cyclohexyl group. In
them, methyl group, ethyl group, etc. are preferred.
[0038] The term "aryl group(s)" as used herein indicates a
monocyclic or bicyclic aromatic substituent(s) composed of 5 to 12
carbon atoms, such as phenyl group, indenyl group, naphthyl group
and fluorenyl group. In them, phenyl group is preferred. The term
"arylthio group" indicates a monocyclic or bicyclic aromatic
substituent(s) composed of 5 to 12 carbon atoms and further
including a thio moiety.
[0039] The term "alkylthio group(s)" as used herein indicates an
alkylthio group(s) having a linear, branched or cyclic alkyl group
having 1 to 12 carbon atoms, preferably 1 to 5 carbon atoms, such
as methylthio group, ethylthio group, n-propylthio group,
isopropylthio group, n-butylthio group, isobutylthio group,
sec-butylthio group, tert-butylthio group, cyclopropylthio group,
cyclobutylthio group, cyclopentylthio group and cyclobutylthio
group.
[0040] The term "acyl group(s)" as used herein indicates a formyl
group, an acyl group(s) having a linear, branched or cyclic alkyl
group having 1 to 6 carbon atoms, acyl group(s) having a linear,
branched or cyclic alkenyl group having 1 to 6 carbon atoms, acyl
group(s) having a linear, branched or cyclic alkynyl group having 1
to 6 carbon atoms or acyl group(s) having an aryl group which may
be substituted, such as formyl group, acetyl group, propionyl
group, butyryl group, isobutyryl group, valeryl group, isovaleryl
group, pivaloyl group, hexanoyl group, acryloyl group, methacryloyl
group, crotonoyl group, isocrotonoyl group, benzoyl group and
naphthoyl group. Acyl groups having a heterocyclic ring can also be
used, for example, furanyl carbonyl group, thienyl carbonyl group,
isoxazolyl carbonyl group and thiazolyl carbonyl group.
[0041] The term "thio acyl group(s)" as used herein indicates a
thio acyl group(s) having a linear, branched or cyclic alkyl group
having 1 to 6 carbon atoms, thio acyl group(s) having a linear,
branched or cyclic alkenyl group having 1 to 6 carbon atoms, thio
acyl group(s) having a linear, branched or cyclic alkynyl group
having 1 to 6 carbon atoms or thio acyl group(s) having an aryl
group which may be substituted, such as formyl group, acetyl group,
propionyl group, butyryl group, isobutyryl group, valeryl group,
isovaleryl group, pivaloyl group, hexanoyl group, acryloyl group,
methacryloyl group, crotonoyl group, isocrotonoyl group, benzoyl
group and naphthoyl group. Thio acyl groups may be incorporated in
a heterocyclic ring, for example, thienyl carbonyl group and
thiazolyl carbonyl group.
[0042] The term "amino acid" refers to an organic acid containing
an amino group. The term includes naturally occurring amino acids
("natural amino acids") such as alanine, valine, leucine,
isoleucine, proline, phenylalanine, tryptophan, methionine,
glycine, serine, threonine, cysteine, asparagine, glutamine,
tyrosine, histidine, lysine, arginine, aspartic acid, and glutamic
acid. Amino acids can be pure L or D isomers or mixtures of L and D
isomers.
[0043] "Prodrugs" refers to compounds, including monomers and
dimers of the compounds of the invention, which have cleavable
groups and become under physiological conditions compounds which
are pharmaceutically active in vivo.
[0044] "Subject" includes humans. The terms "human," "patient" and
"subject" are used interchangeably herein.
[0045] "Therapeutically effective amount" means the amount of a
compound that, when administered to a subject for treating a
disease or disorder, is sufficient to effect such treatment for the
disease or disorder. The "therapeutically effective amount" can
vary depending on the compound, the disease or disorder and its
severity, and the age, weight, etc., of the subject to be
treated.
[0046] "Treating" or "treatment" of any disease or disorder refers,
in one embodiment, to ameliorating the disease or disorder (i.e.,
arresting or reducing the development of the disease or at least
one of the clinical symptoms thereof). In another embodiment
"treating" or "treatment" refers to ameliorating at least one
physical parameter, which may not be discernible by the subject. In
yet another embodiment, "treating" or "treatment" refers to
modulating the disease or disorder, either physically, (e.g.,
stabilization of a discernible symptom), physiologically, (e.g.,
stabilization of a physical parameter), or both. In yet another
embodiment, "treating" or "treatment" refers to delaying the onset
of the disease or disorder, or even preventing the same.
[0047] The present inventors have recently identified the
cystine-glutamate antiporter as a highly novel cellular process
that likely contributes to the pathology underlying schizophrenia.
Importantly, the inventors have collected the first data set
indicating that cysteine prodrugs, used to increase the activity of
cystine-glutamate antiporters, block cognitive deficits and social
withdrawal in the preclinical phencyclidine model of schizophrenia.
Unlike existing medications, cysteine prodrugs appear to exert
antipsychotic properties, in part, by reversing pathology
underlying the disease.
[0048] While no one theory or mechanism of pharmacological effect
is adopted herein, cysteine prodrugs appear to restore diminished
signaling to glutamate receptors and diminished glutathione levels
observed in schizophrenics. A depleted glutathione level can lead
to increased oxidative stress, and impaired cystine-glutamate
antiporter activity, glutamate neurotransmission, synaptic
connection, and gene expression, all of which are observed in
schizophrenia.
[0049] As a related matter, as made evident by the inventors'
findings, impaired cystine-glutamate antiporter activity and faulty
glutamate neurotransmission bear on the issue of uncontrolled drug
use, i.e., drug addiction. Uncontrolled drug use and heightened
susceptibility to relapse are defining features of addiction that
contribute to the transition in drug consumption from a
recreational to a compulsive pattern. Long-term plasticity
resulting in augmented excitatory neurotransmission within
corticostriatal pathways in response to drugs of abuse have been
implicated in addiction. Human cocaine abusers exposed to
craving-inducing stimuli exhibit increased activation of excitatory
circuits originating in cortical regions, including orbital and
prefrontal cortex, and projecting to the ventral striatum; further,
the degree of activation of corticostriatal pathways correlates
with craving in humans.
[0050] Preclinical data also indicate the existence of drug-induced
plasticity leading to activation of corticostriatal pathways.
Activation of these circuits results in heightened extracellular
glutamate in the nucleus accumbens and stimulation of ionotropic
glutamate receptors, both of which are necessary for cocaine primed
reinstatement. Further, the dorsomedial prefrontal cortex has been
shown to be necessary for reinstatement produced by exposure to
drug-paired cues using the contextual reinstatement paradigm and in
response to electrical foot shock. As a result, identification of
cellular mechanisms capable of regulating synaptic glutamate
represent targets in the treatment of addiction.
[0051] Increased excitatory neurotransmission in the nucleus
accumbens may arise, in part, by diminished activity of
cystine-glutamate antiporters. The recent data collected by the
present inventors illustrates that glutamate released from these
antiporters provides endogenous tonic stimulation to group II or
2/3 metabotropic glutamate receptors (mGluRs) and thereby regulates
synaptic glutamate and dopamine release. Thus, altered glutamate
signaling could arise as a consequence of decreased
cystine-glutamate exchange. Repeated cocaine administration has
been shown to blunt the activity of cystine-glutamate exchange,
which likely contributes to a sequence of events, including
diminished group II mGluR autoregulation and increased excitatory
neurotransmission in the nucleus accumbens.
[0052] Cysteine prodrugs, such as N-acetylcysteine ("NAC"), are
used to drive cystine-glutamate exchange by apparently elevating
extracellular cystine levels, thereby creating a steep cystine
concentration gradient. Preclinical studies have shown
N-acetylcysteine to be effective in blocking compulsive
drug-seeking in rodents. Further, extant clinical data also show a
reduction in cocaine use and craving in cocaine abusers receiving
NAC. Unfortunately, the full clinical efficacy of targeting
cystine-glutamate exchange may be unrealized when utilizing NAC due
to extensive first-pass metabolism and limited passive transport of
this drug across the blood-brain barrier. The prodrugs described
and claimed herein will not be significantly eliminated by the
liver and will readily pass the blood-brain barrier. Cysteine is
the reduced form of cystine and is readily oxidized in vivo to
cystine, thus elevating either cysteine or cystine is believed to
increase cystine-glutamate exchange.
[0053] The cysteine prodrug NAC has been previously shown to have a
favorable safety/tolerability profile in human subjects. In fact,
NAC has been used for decades in humans for other indications
(e.g., as a mucolytic, acetaminophen toxicity) and as an
experimental treatment (HIV, cancer) without producing severe
adverse effects. However, NAC undergoes extensive first pass
metabolism requiring the usage of high doses that limit the utility
of the drug and, potentially, increase the chances of side effects
due to the buildup of metabolized by-products. The chemical
entities presently disclosed and claimed herein are designed to
substantially avoid the problem of first pass metabolism and
therefore exhibit increased efficacy as compared to prior cysteine
prodrugs.
##STR00009## ##STR00010##
[0054] The preferred synthetic route to provide cysteine prodrugs
according to the invention will now be described. Scheme 1 depicts
the synthesis of the lead diketopiperazine targets 4 and 5. The
chemistry employed is based on Scholkopf chiral auxiliary chemistry
and provides yields on the kilogram scale. Protection of the thiol
(--SH group) moiety in the cysteine is required to insure the
formation of the Scholkopf chiral auxiliary and prevent other
cyclization reactions. Thiol protection is accomplished by using
either tert-butyl alcohol (in the presence of hydrochloric acid),
phenylsulfenyl chloride or triphenyl methyl chloride (trityl
chloride). Thiol protected cysteine is converted via 2, using the
ethyl ester (methyl ester may also be used) of the desired amino
acid, and undergoes intramolecular cyclization to produce the
prodrug 3. Deprotection of the thiol group produces the lead
diketopiperazine target 4. Depicted in FIG. 1 are exemplary
compounds that can be made using naturally occurring L-amino acids
and D-isomers thereof. Alkylation of carboxyl groups on target 4
produces another prodrug 5 (Scheme 1). Furthermore, the
dealkylation of the carboxyl group on 5 through hydrolysis provides
prodrugs 6a and 6b and eventually 4.
[0055] The synthesis of the symmetrical cystine prodrugs is
preferably accomplished by carrying out the thiol deprotection step
in either a) ethanol with a catalytic amount of mercaptoethanol for
the phenylsulfenyl protected thiol or b) pyridine using a catalytic
amount of iodine for the triphenyl methane protected thiol, as
shown in Scheme 2. However, the free thiol diketopiperazine can be
used to produce symmetrical cystine prodrugs in ethanol and the
presence of a catalytic amount of iodine. An exemplary cystine
prodrug, the cysteine/glycine dimer, is further depicted in Scheme
2.
##STR00011##
[0056] The synthesis of hetero (unsymmetrical) disulfide dimers is
preferably accomplished by using a one-pot reaction with
1-chlorobenzotriazole, as shown in Scheme 3. An alternate method
involves using a catalytic amount of iodine in the presence of an
equal molar amount of any two triphenyl methane protected thiol
cysteine prodrugs. The desired target can be separated and purified
using simple column chromatography.
##STR00012## ##STR00013##
[0057] Unsymmetrical disulfides can be synthesized from any two
sulfide ligands provided by the above-described chemistries.
Accordingly, the invention encompasses symmetrical and
unsymmetrical disulfide dimers synthesized from the combination of
any two sulfide monomers described herein.
[0058] The present method of synthesizing prodrugs according to the
invention has many advantages over previous routes including, but
not limited to: a) same synthetic route leads to both monomers and
dimers (cysteine and cystine prodrugs); b) protection of the thiol
group prevents side (cyclization) reactions; c) the initial monomer
synthesis eliminates problems associated with multiple functional
groups; d) the occurrence of undesired intramolecular and
intermolecular side reactions is decreased; e) and the described
route can be easily expanded to incorporate additional amino
acids.
[0059] Particularly preferred cysteine monomers (prodrugs)
according to the invention are shown in FIG. 1, boldfaced and
underlined. These compounds are preferred either for advantages in
partition coefficients (valine, proline), active transport
(phenylalanine, proline), or breakdown products (cysteine,
glycine). All targets synthesized from the diketopiperazine moiety
are eventually cleaved and/or metabolized by either intra- or
extra-cellular mechanisms to produce cysteine or cystine, which can
then be used in the cystine-glutamate antiporter. FIG. 2 depicts
general chemical formulas for certain cysteine and cystine prodrugs
encompassed by the present invention.
[0060] Accordingly, the invention provides a cysteine prodrug
having the structure:
##STR00014##
wherein: R.sup.1 and R.sup.2 are independently selected from OH,
.dbd.O, or a branched or straight chain C.sub.1 to C.sub.5 alkoxyl
group, with the caveat that when .dbd.O is selected the nitrogen
atom adjacent the carbonyl group thusly formed bears a H and a
single bond joins the adjacent nitrogen to said carbonyl group;
R.sup.3 is H, a branched or straight chain C.sub.1 to C.sub.5
alkyl, a nitrobenzenesulfonyl, a trityl, an aryl thio, an aryl, an
alkylthio, an acyl, a benzoyl, a thio acyl, a thio benzoyl, or a
benzyl group; and R.sup.4 is selected from the side chain groups of
the natural L-amino acids cys, gly, phe, pro, val, ser, arg, asp,
asn, glu, gln, ala, his, ile, leu, lys, met, thr, trp, tyr, or
D-isomers thereof, with the caveat that when R.sup.4 is the side
chain group of the natural L-amino acid gly, R.sup.1 and R.sup.2
are not both selected to be .dbd.O; or a cystine dimer of said
prodrug having the structure:
##STR00015##
wherein: R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are independently
selected from OH, .dbd.O, or a branched or straight chain C.sub.1
to C.sub.5 alkoxyl group, with the caveat that when .dbd.O is
selected the nitrogen atom adjacent the carbonyl group thusly
formed bears a H and a single bond joins the adjacent nitrogen to
said carbonyl group; and R.sup.4 and R.sup.7 are independently
selected from the side chain groups of the natural L-amino acids
cys, gly, phe, pro, val, ser, arg, asp, asn, glu, gln, ala, his,
ile, leu, lys, met, thr, trp, tyr, or D-isomers thereof, with the
caveat that when R.sup.4 and R.sup.7 are both the side chain group
of the natural L-amino acid gly, R.sup.1, R.sup.2, R.sup.5 and
R.sup.6 shall not all be selected to be .dbd.O.
[0061] In certain preferred embodiments, the cysteine prodrug
according to the invention has the structure:
##STR00016##
[0062] The cysteine prodrug may alternatively be provided in the
form of a cystine dimer. Certain preferred cystine dimers according
to the invention have the structure: the form of the cystine dimer
having the structure:
##STR00017##
[0063] The invention provides synthetic routes for the synthesis of
cystine dimers having identical R.sup.4 and R.sup.7 groups or,
alternatively, mixed or non-identical R.sup.4 and R.sup.7 groups.
Cystine dimers of the invention may therefore be of either
symmetric or asymmetric design.
[0064] In certain cysteine prodrugs or cystine dimers of the
invention, at least one R.sup.4 or R.sup.7 group is the side chain
of cysteine and the reactive moiety thereof is further protected by
a branched or straight chain C.sub.1 to C.sub.5 alkyl, a
nitrobenzenesulfonyl, a trityl, an aryl thio, an aryl, an
alkylthio, an acyl, a benzoyl, a thio acyl, a thio benzoyl, or a
benzyl group.
[0065] Upon administration to a subject, compounds according to the
invention pass largely intact through first pass metabolism and
then are hydrolyzed (cleaved) into the corresponding amino acids by
peptidases in cells contained within the CNS. Accordingly, prodrugs
are chemical entities that are readily convertible in vivo to
become pharmaceutically active.
[0066] Scheme 4 and Scheme 5 illustrate yet another approach
provided by the invention in which L-cysteine is protected as acyl
analogs with alkyl esters to improve the partition coefficient (Log
P) and circulatory half life in the blood to provide improved
passive delivery into the brain through the blood brain
barrier.
##STR00018##
[0067] In Scheme 5, glycine is incorporated into some of the
protected cysteine analogs (17) to provide a more efficient method
of delivery of both amino acids. Various alkyl alcohols are
incorporated into targets from Scheme 4 (12). Symmetrical cystine
targets are synthesized from the corresponding cysteine analogs by
the addition of a catalytic amount of iodine. Again, all prodrugs
are hydrolyzed (cleaved) into the active corresponding amino acid
in vivo. The molecules described in Scheme 4 and Scheme 5 result in
more exposure and increased brain levels as compared to previous
versions. It is noteworthy that this approach alters the partition
coefficient by completely protecting the cysteine/cystine moiety.
Synthetic challenges, such as solubility and stability of resulting
intermediates and targets, have previously prevented others in the
field from obtaining protected products in significant quantities,
even for research studies. FIG. 3 depicts general chemical formulas
for certain protected cysteine analogs encompassed by the present
invention.
##STR00019##
[0068] Accordingly, the invention further encompasses protected
cysteine analogs having the structure:
##STR00020##
or a cystine dimer of the protected cysteine analog having the
structure:
##STR00021##
[0069] wherein R.sup.1 through R.sup.6 are independently selected
from a branched or straight chain C.sub.1 to C.sub.5 alkyl, a
phenyl, or a benzyl group.
[0070] Preferable protected cysteine analogs according to the
invention have the structure:
##STR00022##
[0071] Alternatively, protected cysteine analogs may be provided in
the form of the corresponding cystine dimers. Certain preferred
cystine dimers have the structures:
##STR00023##
[0072] Relative to the protected cysteine analogs, the invention
further provides a method of reducing schizophrenia in a subject by
administering to a subject an effective amount of a protected
cysteine analog or cystine dimer thereof according to the
invention, whereby schizophrenia is reduced in said subject.
Administration is preferably via the oral route.
[0073] Of course, the invention further encompasses pharmaceutical
compositions containing a protected analog or dimer thereof in
combination with a pharmaceutically-acceptable carrier. Methods of
formulating/manufacturing such pharmaceutical compositions for the
treatment of schizophrenia or for reducing drug craving in a
subject are also within the invention's scope.
[0074] In certain preferred embodiments, the compounds of the
invention will be provided as pharmaceutically acceptable salts.
Other salts may, however, be useful in the preparation of the
compounds according to the invention or of their pharmaceutically
acceptable salts. Suitable pharmaceutically acceptable salts of the
compounds of this invention include acid addition salts which may,
for example, be formed by mixing a solution of the compound
according to the invention with a solution of a pharmaceutically
acceptable acid such as hydrochloric acid, sulphuric acid,
methanesulphonic acid, fumaric acid, maleic acid, succinic acid,
acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid,
carbonic acid or phosphoric acid. Furthermore, where the compounds
of the invention carry an acidic moiety, suitable pharmaceutically
acceptable salts thereof may include alkali metal salts, e.g.
sodium or potassium salts, alkaline earth metal salts, e.g. calcium
or magnesium salts; and salts formed with suitable organic ligands,
e.g. quaternary ammonium salts.
[0075] Where the compounds according to the invention have at least
one asymmetric center, they may accordingly exist as enantiomers.
Where the compounds according to the invention possess two or more
asymmetric centers, they may additionally exist as
diastereoisomers. It is to be understood that all such isomers and
mixtures thereof in any proportion are encompassed within the scope
of the present invention.
[0076] The invention also provides pharmaceutical compositions
comprising one or more compounds of this invention in association
with a pharmaceutically acceptable carrier. Preferably these
compositions are in unit dosage forms such as tablets, pills,
capsules, powders, granules, sterile parenteral solutions or
suspensions, metered aerosol or liquid sprays, drops, ampoules,
auto-injector devices or suppositories; for oral, parenteral,
intranasal, sublingual or rectal administration, or for
administration by inhalation or insufflation. It is also envisioned
that the compounds of the present invention may be incorporated
into transdermal patches designed to deliver the appropriate amount
of the drug in a continuous fashion.
[0077] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutically
acceptable carrier, e.g. conventional tableting ingredients such as
corn starch, lactose, sucrose, sorbitol, talc, stearic acid,
magnesium stearate, dicalcium phosphate or gums, and other
pharmaceutical diluents, e.g. water, to form a solid preformulation
composition containing a homogeneous mixture for a compound of the
present invention, or a pharmaceutically acceptable salt thereof.
When referring to these preformulation compositions as homogeneous,
it is meant that the active ingredient is dispersed evenly
throughout the composition so that the composition may be easily
subdivided into equally effective unit dosage forms such as
tablets, pills and capsules. This solid pre-formulation composition
is then subdivided into unit dosage forms of the type described
above containing from 0.1 to about 500 mg of the active ingredient
of the present invention. Typical unit dosage forms contain from 1
to 100 mg, for example, 1, 2, 5, 10, 25, 50 or 100 mg, of the
active ingredient. The tablets or pills of the novel composition
can be coated or otherwise compounded to provide a dosage affording
the advantage of prolonged action. For example, the tablet or pill
can comprise an inner dosage and an outer dosage component, the
latter being in the form of an envelope over the former. The two
components can be separated by an enteric layer which, serves to
resist disintegration in the stomach and permits the inner
component to pass intact into the duodenum or to be delayed in
release. A variety of materials can be used for such enteric layers
or coatings, such materials including a number of polymeric acids
and mixtures of polymeric acids with such materials as shellac,
cetyl alcohol and cellulose acetate.
[0078] The liquid forms in which the novel compositions of the
present invention may be incorporated for administration orally or
by injection include aqueous solutions, suitably flavored syrups,
aqueous or oil suspensions, and flavored emulsions with edible oils
such as cottonseed oil, sesame oil, coconut oil or peanut oil, as
well as elixirs and similar pharmaceutical vehicles. Suitable
dispersing or suspending agents for aqueous suspensions include
synthetic and natural gums such as tragacanth, acacia, alginate,
dextran, sodium caboxymethylcellulose, methylcellulose,
polyvinylpyrrolidone or gelatin.
[0079] The compounds according to the present invention exhibit
schizophrenia reducing/alleviating activity, as demonstrated by
standard protocols. For example, efficacy of the present inventive
compounds in the schizophrenia context has been demonstrated by
assaying startle response to a load stimulus (pulse) when preceded
by a pre-pulse stimulus. Accordingly, another aspect of the
invention provides a method for the reduction of schizophrenia in a
subject in need of such treatment by administration of an effective
amount of compound according to the invention or a precursor
thereof. In the treatment of schizophrenia, suitable dosage level
(i.e, an effective amount) is about (1-5000) mg/kg, per day,
preferably about (30-3000) mg/kg per day, and especially about
(50-1000) mg/kg per day. The compounds may be administered on a
regimen of 1 to 4 times per day, or on a continuous basis.
[0080] Accordingly, the present invention further provides a method
of reducing schizophrenia in a subject. Such a method includes
steps of administering to the subject an effective amount of a
cysteine prodrug or cystine dimer thereof according to the
invention, whereby schizophrenia is reduced in the subject.
Administration is preferably accomplished by oral delivery.
[0081] As well, the compounds according to the present invention
may also exhibit the ability to reduce drug cravings. This
desirable activity can be shown in animal models involving
drug-seeking behavior produced by stress, drug-paired cues, or a
cocaine priming injection. Accordingly, yet another aspect of the
invention is directed to a method of reducing a drug craving in a
subject in need thereof. Such a method includes the step of
administering an effective amount of a compound having the chemical
structure of compound according to the invention, or a precursor
thereof, to the subject whereby the drug craving is reduced in the
subject. In the treatment of drug cravings, suitable dosage level
(i.e., effective amount) is about (1-5000) mg/kg, per day,
preferably about (30-3000) mg/kg per day, and especially about
(50-1000) mg/kg per day.
[0082] The invention therefore provides a method of reducing drug
craving in a subject. Such a method includes steps of administering
to the subject an effective amount of a cysteine prodrug or cystine
dimer of the invention, whereby drug craving is reduced in the
subject. Again, administration is preferably via the oral
route.
[0083] The following examples are, of course, offered for
illustrative purposes only, and are not intended to limit the scope
of the present invention in any way. Indeed, various modifications
of the invention in addition to those shown and described herein
will become apparent to those skilled in the art from the foregoing
description and the following examples and fall within the scope of
the appended claims.
[0084] In the following examples, the compounds where named based
on the following criteria: Cysteine prodrugs (monomers) were
assigned names as (Assigned number from Scheme--Amino acid
incorporated; a=glycine, b=phenylalanine, c=proline, d=valine,
e=cysteine) (i.e. 3c-a: Target 3c from Scheme 1 with glycine
incorporated) or alternatively (Assigned number from Scheme with a
"letter" indicating the amino acid incorporated) (i.e. 4a: Target 4
from Scheme 1 with glycine incorporated), Cystine prodrugs were
named as (Assigned number from Scheme with a "letter" indicating
the amino acid incorporated) (i.e. 7b: Target 7 from Scheme 2 with
phenylalanine incorporated) or alternatively as (Assigned number
from Scheme with a "letter" indicating the amino acid
incorporated--dimer) (i.e. 5a-dimer: The dimer of Target 5 from
Scheme 1 with glycine incorporated). Unsymmetrical Cystine prodrugs
were named as (Assigned number from Scheme--Amino acid incorporated
(monomer 1)--Amino acid incorporated (monomer 2)) (i.e. 11-a-b:
Target 11 from Scheme 3 with glycine incorporated into monomer 1
and phenylalanine incorporated into monomer 2).
EXAMPLES
Example 1
Experimental for Scheme 1 Compounds
##STR00024##
[0086] Preparation of p-Tolyl hypochlorothioite: Under a nitrogen
atmosphere, N-chloro-succinimide (48.1 g, 0.36 mole) was slurried
in 200 ml of methylene chloride. While stirring at room
temperature, 4-methylbenzenethiol (29.8 g, 0.24 mole) was added; (2
g initial addition to start reflux and the remainder at a rate to
maintain reflux approximate 10 min.) The clear solution which
resulted was then stirred at room temperature for 30 minutes. A
small amount of precipitate which formed was removed by filtration.
The filtrate, assumed to contain the theoretical quantity of
4-methylbenzenesulfenyl chloride (38.1 g, 0.24 mole), was used
immediately and directly in the next step. Alternatively,
4-methylbenzene-sulfenyl chloride was isolated by evaporation to an
solid to its further use.
[0087] (R)-2-amino-3-(phenyldisulfanyl)propanoic acid (1b): To a
solution of L-cysteine hydrochloride mono-hydrate (47 g, 0.3 mol)
in absolute ethanol (900 mL) was added powdered sodium bicarbonate
(30 g, 0.36 mol) at 0.degree. C. in one portion. Phenylsulfenyl
chloride (50 g, 0.345 mol) was added dropwise with stirring to the
mixture. After the complete addition of the reagent, the reaction
mixture was allowed to stand at room temperature and the sodium
chloride which was produced during the reaction was removed by
filtration. After basifying the mixture by the addition of pyridine
(38 mL) into the filtrate, the fine precipitate which formed was
allowed to stand for a couple of hours, then filtrated and washed
well with ethanol and dried to provide the crude product as a white
solid. After recrystallization from aqueous HCl (0.5 N, 4000 mL),
the final product S-thiol-phenyl-L-cysteine (1b) was obtained (52
g) in 76% yield as colorless plates. 1b: m.p. 192.degree. C.
(decomp). .sup.1H NMR (300 MHz, CD.sub.3CO.sub.2D): .delta.
3.53-3.76 (m, 2H), 4.89 (t, 1H), 7.26-7.88 (m, 5H); .sup.13C NMR
(75.5 MHz, CD.sub.3CO.sub.2D): .delta. 35.5, 52.5, 127.6, 128.5,
129.1, 129.3, 133.5, 171.6. This material was employed directly in
the next step.
##STR00025##
[0088] 2-Amino-3-tritylsulfanyl-propionic acid
(S-Trityl-L-cysteine) (1c): L-Cysteine hydro-chloride (100 g, 0.634
mol) and trityl chloride (270 g, 0.969 mol) were stirred in DMF
(400 mL) for 2 days at room temperature. A 10% sodium acetate
solution (3.5 L) was then added dropwise and the white precipitate
which formed was filtered and washed with distilled water.
Afterward, the residue was stirred in acetone at 50.degree. C. for
30 min after which it was cooled to 0.degree. C. and filtered. The
precipitate was washed with a little acetone and diethyl ether and
dried in vacuo. S-Trityl-L-cysteine 1c (205 g, 89%) was obtained as
a white powder. 1c: m.p. 192.degree. C. (decomp); .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 2.45 (dd, 1H, J=9 Hz, 12 Hz), 2.58 (dd,
1H, J=4.4 Hz, 12 Hz), 2.91 (m, 1H), 7.22-7.36 (m, 15H); .sup.13C
NMR (75.5 MHz, DMSO-d.sub.6): .delta. 33.8, 53.7, 66.4, 127.1,
127.8, 128.1, 128.4, 129.5, 144.5, 168.4. This material was
directly used in the next step without further purification.
##STR00026##
[0089] (R)-4-((phenyldisulfanyl)methyl)oxazolidine-2,5-dione (2b):
To a rapidly stirred (over-head stirrer) suspension of
S-thiol-phenyl-L-cysteine (1b) (57.5 g, 0.25 mol) in THF (250 mL)
was added solid triphosgene (26 g, 88 mmol) in one portion at
45-50.degree. C. (before addition, remove the heating mantle). When
the temperature drops to 45.degree. C., put the heating mantle back
on and maintain the inside temperature around 45-50.degree. C.
until the solution becomes homogeneous. After the removal of the
heating mantle, the solution was purged with argon overnight into a
NaOH bubbler to remove any residual phosgene. The solvent was
evaporated in vacuo and this provided anhydride 2b (55 g) in 85%
yield. 2b: m.p. 217.degree. C. (decomp); .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 2.90-2.98 (m, 1H), 3.30 (d, 1H, J=12 Hz), 4.68
(d, 1H, J=9 Hz), 6.01 (s, 1H), 7.34-7.58 (m, 5H); .sup.13C NMR
(75.5 MHz, CD.sub.3Cl.sub.3): .delta. 39.4, 56.5, 128.3, 128.9,
129.5, 135.2, 150.8, 167.7. Due to the unstable nature of this
anhydride, it was stored in the refrigerator overnight under an
atmosphere of argon and used immediately the next day without
further purification.
##STR00027##
[0090] 4-Tritylsulfanylmethyl-oxazolidine-2,5-dione (2c) was
prepared following the procedure for preparation of 2b as a brown
oil in 85% yield. 2c: .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
2.70-2.85 (m, 2H), 3.47-3.56 (m, 1H), 5.62 (s, 1H), 7.07-7.73 (m,
15H). This material was directly used in the next step without
further purification.
Representative Procedure for Synthesis of Diketopiperazine
Targets:
##STR00028##
[0092] 2,5-Piperazinedione, 3-(mercaptomethyl)-(4a): a). A solution
of the N-carboxy-anhydride 2b (35.7 g, 0.14 mol) in THF (160 mL)
was added dropwise to a vigorously stirred (overhead stirrer)
mixture of glycine ethyl ester hydrochloride (28 g, 0.16 mol),
freshly distilled triethylamine (20.4 g, .about.28 mL, 0.20 mol)
and dry chloroform (240 mL) at -78.degree. C. in a three-neck flask
(2 L). The reaction mixture was allowed to warm to 0.degree. C.
over 8 h, and then was stirred at rt for 12 h, after which the
reaction solution was filtered to remove the triethylamine
hydrochloride which precipitated. The filtrate was then
concentrated under reduced pressure (<40.degree. C.) and the
crude dipeptide ester was used for the preparation of the
diketopiperazine 4a without further purification. .sup.1H NMR (300
MHz, CDCl.sub.3): .delta. 1.29 (t, 3H), 1.93 (br, 2H), 2.74-2.82
(m, 1H), 3.40 (dd, 1H), 3.73 (dd, 1H), 4.03-4.19 (m, 2H), 4.19-4.26
(m, 2H), 7.34-7.58 (m, 5H). b). The crude dipeptide ester (37.6 g,
0.12 mol) was heated in refluxing toluene (1000 mL) for 12 h and
then cooled down to rt and kept at 0.degree. C. for 16 h. The
bislactam 4a which precipitated was isolated by vacuum filtration,
washed with ether (3.times.150 mL), and dried under vacuum at
100.degree. C. to provide pure diketopiperazine 4a (10.0 g) in 45%
yield. The resulting filtrate produced from washing the desired
diketopiperazine was evaporated under vacuum and toluene (800 mL)
was added to the residue. The toluene solution was heated at reflux
for another 40 h (under argon) and then the above steps were
repeated to collect another 5-8 grams of diketopiperazine 4a
(combined yield, 73%). 4a: m.p. 258.degree. C.; .sup.1H NMR (300
MHz, DMSO-d.sub.6): .delta. 3.09-3.26 (m, 2H), 3.68-3.88 (m, 2H),
4.10 (s, 1H), 8.17 (s, 1H), 8.19 (s, 1H); .sup.13C NMR (500 MHz,
DMSO-d.sub.6): .delta. 43.5, 44.7, 54.3, 166.2, 166.6; EIMS (m/e,
relative intensity) 160 (M.sup.+, 12), 140(5), 126(72), 114(100),
97(20), 85 (30).
[0093] 3-Phenyldisulfanylmethyl-piperazine-2,5-dione (3b-a): c).
The solution which resulted from step b above was cooled to
0.degree. C. and keep at 0.degree. C. for 12 h. The precipitate
which resulted was filtered and provided phenyl-thiol analog 3b-a
in 30% yield. 3b-a: .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta.
3.09-3.21 (m, 2H), 3.65-3.82 (m, 2H), 4.10 (s, 1H), 7.11-7.55 (m,
5H), 8.18 (s, 1H), 8.20 (s, 1H); .sup.13C NMR (75.5 MHz,
DMSO-d.sub.6): .delta. 43.5, 47.8, 54.2, 125.6, 127.7, 128.2,
129.5, 166.2, 166.6; EIMS (m/e, relative intensity) 268 (M.sup.+,
55), 250(35), 218(68), 159(66), 141(80), 126 (70).
##STR00029##
[0094] (3R,6R)-3-benzyl-6-(mercaptomethyl)piperazine-2,5-dione
(4b): was prepared in 75% yield following the procedure for
preparation of 4a and obtained as a light yellow solid. 4b: m.p.
>265.degree. C. (decomp.); .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 1.26 (d, J=6.99 Hz, 1H), 3.05-3.49 (m, 2H), 3.66-3.89 (m,
3H), 4.10 (s, 1H), 7.13-7.31 (m, 5H), 8.23 (s, 1H), 8.28 (s, 1H);
.sup.13C NMR (75.5 MHz, CDCl.sub.3) .delta. 19.0, 37.9, 44.7, 48.1,
51.2, 54.4, 126.5, 129.1, 129.4, 165.9, 166.5. EIMS (m/e, relative
intensity) 250 (M.sup.+, 10), 216(12), 160(5), 113(11), 91
(100).
##STR00030##
[0095] (6R)-3-isopropyl-6-(mercaptomethyl)piperazine-2,5-dione
(4d): was prepared in 74% yield following the procedure for
preparation of 4a and obtained as a white solid. 4d: m.p.
>275.degree. C.; .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
0.84 (dd, J=7.14, 6.63 Hz, 3H), 0.94 (dd, J=8.07, 6.9 Hz, 3H),
2.17-2.20 (m, 1H), 3.07-3.18 (m, 2H), 3.73 (s, 1H), 4.22 (s, 1H),
8.12 (s, 1H), 8.18s (s, 1H); .sup.13C NMR (75.5 MHz, CDCl.sub.3)
.delta. 17.5, 18.8, 42.9, 53.9, 59.7, 166.7, 167.2; HRMS m/z
C.sub.10H.sub.18N.sub.2O.sub.2S.sub.2(M-H).sup.+ calcd 201.0698,
found 201.0691.
##STR00031##
[0096]
(6R)-3-(tert-butylthiomethyl)-6-(mercaptomethyl)piperazine-2,5-dion-
e (4e): was prepared in 70% yield following the procedure for
preparation of 4a and obtained as a yellow solid. 4e: m.p.
>280.degree. C. (decomp.); .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 1.25 (s, 9H), 2.88-2.92 (m, 1H), 3.03-3.10 (q, J=7.5 Hz,
1H), 3.18-3.21 (m, 1H), 3.51 (d, J=14.4 Hz, 1H), 4.14 (s, 2H), 8.13
(s, 1H), 8.24 (s, 1H); .sup.13C NMR (75.5 MHz, CDCl.sub.3) .delta.
31.1, 32.1, 43.2, 47.8, 54.1, 54.9, 166.3, 170.8; EIMS (m/e,
relative intensity) 262 (M.sup.+, 30), 228(40), 206(45), 173(50),
160(70), 126 (100); HRMS m/z C.sub.10H.sub.18N.sub.2O.sub.2S.sub.2
(M+H).sup.+ calcd 263.0482, found 263.0489.
##STR00032##
[0097]
(3R,8aR)-3-((phenyldisulfanyl)methyl)hexahydropyrrolo[1,2-a]pyrazin-
e-1,4-dione (3b-c) was prepared in 82% yield following the
procedure for preparation of 3b-a and obtained as a yellow solid.
3b-c: m.p. 120.degree. C.; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 1.66-2.02 (m, 1H), 2.03-2.11 (m, 2H), 2.36 (m, 1H),
2.80-2.89 (m, 1H), 3.54-3.62 (m, 3H), 4.07-4.10 (m, 1H), 4.39 (dd,
J=1.83, 1.77 Hz, 1H), 6.35 (s, 1H), 7.28-7.57 (m, 5H); .sup.13C NMR
(75.5 MHz, CDCl.sub.3) .delta. 22.5, 28.2, 38.5, 45.4, 53.3, 59.1,
127.8, 128.6, 129.2, 135.6, 164.3, 169.0.
##STR00033##
[0098] 3-Tritylsulfanylmethyl-piperazine-2,5-dione (3c-a) was
prepared following the similar procedure for preparation of 4a.
3c-a: m.p. 225-227.degree. C. [.alpha.].sub.D.sup.26=+7.8.degree.
(c=1.05, CHCl.sub.3). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
2.73-2.91 (m, 2H), 3.12 (d, 1H, J=12.3 Hz), 3.95 (s, 1H), 5.80 (s,
1H), 5.82 (s, 1H), 7.20-7.62 (m, 15H). .sup.13C NMR (75.5 MHz,
CDCl.sub.3): .delta. 35.9, 44.8, 53.0, 126.9, 128.1, 129.4, 144.0,
166.6. This material was directly used in the next step without
further purification.
Representative Procedure for Synthesis of Dialkylated
Diketopiperazine:
##STR00034##
[0099] (3,6-Diethoxy-2,5-dihydro-,pyrazin-2-yl)-methanethiol
(5a)
[0100] Preparation of Triethyloxonium Tetrafluoroborate: (Note:
Triethyloxonium tetra-fluoroborate is an expensive reagent;
however, it is relatively easy to prepare even on large scale). A
three-neck flask (500 mL), pressure equilibrating dropping funnel
(125 mL) and a condenser were dried in an oven at 150.degree. C.
and assembled while hot under an atmosphere of argon. When the
equipment had cooled to rt, ether [(100 mL) which had been
previously dried over sodium benzophenone ketyl] and boron
trifluoride diethyletherate (91 g, .about.87 mL, 64 mmol) were
combined [Note: On this scale the colorless BF.sub.3 etherate was
obtained from a freshly opened new bottle. If the reagent was
slightly yellow or if the reaction was scaled down, the BF.sub.3
etherate needed to be vacuum distilled first]. The ethereal
solution which resulted was heated to a gentle reflux after which
dry epichlorohydrin (48.8 g, 41 mL, 51.8 mmol) was added dropwise
over 1 h. The mixture was heated at reflux for an additional 1 h
and allowed to stand at rt (under argon) overnight. The ether was
removed by applying a positive pressure of argon in one neck of the
flask while forcing the ether out through a filter stick (fritted
glass tube) inserted into another neck of the flask and into a
collection flask. The slightly yellow solid which remained in the
flask was rinsed twice in the same manner with anhydrous ether
(3.times.50 mL) to provide a crystalline white solid. The solid was
not weighed but directly used in the next step. The following
sequence was based on the yield of this reaction process at the
level of 80-85%.
[0101] Dry CH.sub.2Cl.sub.2 (100 mL) was added to the flask (500
mL) which contained the freshly prepared triethyloxonium
tetrafluoroborate (.about.42 g, 336 mmol) from the previous
reaction (under argon). To this solution was added the
diketopiperazine 4a (5 g, 31.2 mmol) in portions with stirring
(overhead stirrer). After 2 h the reaction mixture became
homogenous. The solution was stirred at rt under argon for 72 h
after which the mixture was added via a cannula to an aq solution
of NH.sub.4OH (14%, 100 mL) mixed with ice (100 g). The organic
layer was washed with a saturated aq solution of NaHCO.sub.3
(2.times.50 mL) and brine (80 mL) after which it was dried
(K.sub.2CO.sub.3). After filtration the solvent was removed under
reduced pressure to provide the bis-ethoxy lactim ether 5a as a
clear yellow liquid that was further purified by flash
chromatography (EtOAc:Hexane=1:4) in 71% yield (4.8 g, 22 mmol).
5a: [.alpha.].sub.D.sup.26=+52.2.degree. (c=2.5, CHCl.sub.3).
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.32-1.36 (m, 6H),
3.27-3.30 (m, 3H), 4.08-4.22 (m, 6H), 4.39 (s, 1H); .sup.13C NMR
(75.5 MHz, CDCl.sub.3): .delta. 14.7, 46.3, 47.5, 56.1, 61.5, 61.6,
162.7, 163.6; HRMS m/z C.sub.9H.sub.16N.sub.2O.sub.2S (M+H).sup.+
calcd. 217.2982, found 217.2990.
##STR00035##
[0102]
(3R,6R)-6-Benzyl-5-ethoxy-3-(ethylthiomethyl)-1,6-dihydropyrazin-2(-
3H)-one (6b-b) was prepared in 30% yield following the procedure
for preparation of 5b using only 1 equiv. of triethyloxonium
tetrafluoroborate and obtained as a yellow solid. 6b-b: m.p.
118.degree. C.; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.19 (t,
J=7.41 Hz, 3H), 1.30 (t, J=7.08 Hz, 3H), 2.37-2.45 (m, 3H),
2.82-3.01 (m, 1H), 2.95 (d, J=3.09 Hz, 1H), 3.03 (d, J=3.12 Hz,
1H), 3.23 (q, J=32.6, 5.1 Hz, 2H), 4.14-4.19 (m, 2H), 4.46-4.47 (m,
1H), 6.19 (s, 1H), 7.17-7.29 (m, 5H); .sup.13C NMR (75.5 MHz,
CDCl.sub.3): .delta. 14.i, 14.5, 25.7, 35.4, 39.8, 50.6, 60.0,
61.6, 126.5, 127.8, 130.2, 136.7, 157.8, 170.1; HRMS m/z
(M+H).sup.+ calcd. 305.1515, found 305.1522.
Example 2
Representative Procedure for Synthesis of Bis-Dipiperazinedione
##STR00036##
[0104] Bis[2,5-Piperazinedione, 3-(mercaptomethyl)-] (7a): The
trityl protected diketo-piperazine 3c-a (1.5 g, 3.73 mmol) was
dissolved in a solution of methylene chloride (20 mL) and methanol
(40 mL) with stirring. Pyridine (1.2 mL, 15 mmol) was then added to
the resulting mixture, followed by a solution of iodine (0.97 g,
3.8 mmol) in methanol (5 mL). The mixture was allowed to stir for 1
h at room temperature. No precipitate had formed by this time;
however, TLC analysis indicated that the reaction was proceeding
slowly by the appearance of a new spot under the starting material
(UV light). A precipitate began to form within 2 h after
concentrating the solution to a volume of 10 mL and methanol (30
mL) was added to result in a total volume of 40 mL. The solution
was stirred an additional 23 h and the precipitate was filtered
off. The solid was washed with cold methanol and then decolorized
by shaking with 10% aqueous sodium bisulfite (10 mL). The
precipitate was filtered and dried to yield dimer 7a as white solid
(680 mg, 57%). 7a: m.p. >300.degree. C. .sup.1H NMR (300 MHz'
DMSO-d.sub.6) .delta. 3.11-3.21 (m, 2H), 3.70 (d, 1H, J=0.96 Hz),
3.73 (d, 1H, J=0.99 Hz), 4.11 (s, 1H), 8.17 (s, 1H), 8.19 (s, 1H);
.sup.13C NMR (75.5 MHz, DMSO-d.sub.6): .delta. 44.0, 45.2, 54.8,
166.7, 167.1; HRMS m/z (M+H).sup.+ calcd. 319.0535, found
319.0533.
##STR00037##
[0105]
(3S,6S)-3-Benzyl-6-(((((2R,5R)-5-benzyl-3,6-dioxopiperazin-2-yl)met-
hyl)disulfanyl)methyl)piperazine-2,5-dione (7b): was prepared in
63% yield following the procedure for preparation of 7a and
obtained as a yellow solid. 7b: m.p. >280.degree. C. (decomp.);
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.29 (s, 9H), 2.85-2.92
(m, 2H), 3.10-3.13 (m, 2H), 4.14 (s, 2H), 8.12 (s, 2H); .sup.13C
NMR (75.5 MHz, CDCl.sub.3): .delta.31.1, 32.1, 42.5, 43.2, 53.9,
54.1, 166.2, 166.3.
##STR00038##
[0106]
(3R,3'R,6R,6'R)-6,6'-disulfanediylbis(methylene)bis(3-isopropylpipe-
razine-2,5-dione) (7d): was prepared in 65% yield following the
procedure for preparation of 7a and obtained as a white solid. 7d:
m.p. 270.degree. C.; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.86
(d, J=6.75 Hz, 3H), 0.96 (d, J=7.05 Hz, 3H), 2.17-2.21 (m, 1H),
3.07-3.19 (m, 2H), 3.72 (s, 1 h), 4.33 (s, 1H), 8.11 (s, 1H), 8.17
(s, 1H); .sup.13C NMR (75.5 MHz, CDCl.sub.3): .delta. 17.5, 18.8,
31.4, 42.9, 53.9, 59.7, 166.7, 167.2; HRMS m/z (M+H).sup.+ calcd.
403.1474, found 403.1479.
##STR00039##
[0107]
(3R,6S)-3-(tert-Butylthiomethyl)-6-(((((2R,5S)-5-(tert-butylthiomet-
hyl)-3,6-dioxo-piperazin-2-yl)methyl)disulfanyl)methyl)piperazine-2,5-dion-
e (7e): was prepared in 65% yield following the procedure for
preparation of 7a and obtained as a yellow solid. 7e: m.p.
278.degree. C.; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.29 (s,
9H), 2.85-2.92 (m, 2H), 3.10-3.13 (m, 2H), 4.14 (s, 2H), 8.12 (s,
2H); .sup.13C NMR (75.5 MHz, CDCl.sub.3): .delta. 31.1, 32.1, 42.5,
43.2, 53.9, 54.1, 166.2, 166.3.
Representative Procedure for Synthesis of
Bis[(3,6-Diethoxy-2,5-Dihy-Dro-Pyrazin-2-yl)-methanethiol]
(5a-dimer):
##STR00040##
[0108] To the bis-ethoxy lactim ether 5a (400 mg, 1.85 mmol) in dry
EtOH (10 mL) was added a catalytic amount of I.sub.2 (50 mg, 10%
mmol) at rt. The mixture was stirred for 6.about.12 h under air
until the analysis (TLC, silica gel) indicated the reaction was
complete (new spot appeared under S.M. on the TLC plate). The
organic solvent was evaporated under reduced pressure. The mixture
which resulted was dissolved into EtOAc (20 mL), washed with sat.
sodium thiosulfate (5-10 mL) and dried (Na.sub.2SO.sub.4). The
solvent was then removed under reduced pressure which provided the
dimer 5a-dimer: .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.32-1.36
(m, 6H), 3.27-3.30 (m, 3H), 4.08-4.22 (m, 6H), 4.39 (s, 1H);
.sup.13C NMR (75.5 MHz, CDCl.sub.3): .delta. 14.7, 46.3, 47.5,
56.1, 61.5, 61.6, 162.7, 163.6; The NMR spectra was identical to
its monomer except the S--H bond had disappeared. HRMS m/z
(M+H).sup.+ calcd. 431.1787, found 431.1790.
##STR00041##
[0109]
1,2-Bis(((2R,5R)-5-benzyl-3,6-diethoxy-2,5-dihydropyrazin-2-yl)meth-
yl)disulfane (5b-dimer): was prepared in 65% yield following the
procedure for preparation of 5a-dimer and obtained as a yellow
liquid. 5b-dimer: .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
1.26-1.35 (m, 6H), 2.45-2.57 (m, 1H), 3.05-3.22 (m, 2H), 3.50-3.82
(m, 1H), 4.07-4.18 (m, 5H), 4.32-4.38 (m, 1H), 7.06-7.28 (m, 5H);
.sup.13C NMR (75.5 MHz, CDCl.sub.3): .delta. 14.3, 39.6, 42.9,
43.0, 54.9, 57.1, 60.7, 60.8, 126.2, 126.5, 127.8, 137.0, 162.2,
630024.00008 162.6; The NMR spectra was identical to its monomer
except the S--H bond had disappeared. HRMS m/z (M+H).sup.+ calcd.
611.2681, found 611.2677.
##STR00042##
[0110]
1,2-Bis(((2R,5R)-3,6-diethoxy-5-isopropyl-2,5-dihydropyrazin-2-yl)m-
ethyl)disulfane (5c-dimer): was prepared in 60% yield following the
procedure for preparation of 5a-dimer and obtained as a colorless
liquid. 5c-dimer: .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
0.76-0.78 (m, 3H), 1.06-1.09 (m, 3H), 1.25-1.31 (m, 6H), 2.18-2.23
(m, 1H), 2.82-3.01 (m, 1H), 3.21-3.45 (m, 1H), 3.54-3.70 (m, 2H),
4.07-4.33 (m, 4H); .sup.13C NMR (75.5 MHz, CDCl.sub.3): .delta.
14.2, 17.3, 31.1, 31.7, 45.2, 55.3, 60.5, 60.7, 161.0, 163.1; HRMS
m/z (M+H).sup.+ calcd. 515.2726, found 515.2731.
Example 3
Alternative Route for Synthesis of Asymmetric
Bis-Dipiperazinedione
##STR00043##
[0112] Bis[2,5-Piperazinedione, 3-(mercaptomethyl)-](11-a-b): The
trityl protected diketo-piperazine 3c-a (246 mg, 0.5 mmol) and 3c-b
(201 mg, 0.5 mmol) were dissolved in a solution of methylene
chloride (5 mL) and methanol (10 mL) with stirring. Pyridine (0.3
mL, 3.75 mmol) was then added to the resulting mixture, followed by
a solution of iodine (126 mg, 0.5 mmol) in methanol (3 mL). The
mixture was allowed to stir for 1 h at room temperature. No
precipitate had formed by this time; however, TLC analysis
indicated that the reaction was proceeding slowly by the appearance
of a new spot under the starting material (UV light). A precipitate
began to form within 2 h after concentrating the solution to a
volume of 2 mL and methanol (5 mL) was added to result in a total
volume of 10 mL. The solution was stirred an additional 23 h and
the precipitate was filtered off. The solid was washed with cold
methanol. The precipitate was filtered and dried to yield dimer
11-a-b as yellow solid (120 mg, 60%). 11-a-b: .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 2.89-2.91 (m, 2H), 3.09-3.21 (m, 3H),
3.33-3.87 (m, 4H), 4.11 (s, 1 H), 4.21 (s, 1H), 7.13-7.36 (m, 5H),
8.07 (s, 1H), 8.32 (s, 2H), 8.58 (s, 1H); .sup.13C NMR (75.5 MHz,
DMSO-d.sub.6) .delta. 42.3, 42.6, 43.1, 44.7, 53.3, 54.2, 54.3,
55.8, 127.2, 128.2, 130.6, 136.4, 165.9, 166.1, 166.5.
Example 4
Experimental for Scheme 4 and 5 Compounds
Representative Procedure for Synthesis of Protected Analogs
(16)
##STR00044##
[0114] N,S-Dibenzoyl-L-cysteine Ethyl Ester (16): To a solution of
pure L-cysteine ethyl ester hydrochloride (7.5 g, 40 mmol) in
pyridine (30 mL) precooled at 0.degree. C., benzyol chloride (10
mL) was added. After being kept for 1 h at room temperature, the
mixture was poured onto ice. The precipitate was collected by
filtration and was recrystallized from methanol in 88% yield (12
g). 16: m.p. 81.degree. C.; .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. 1.41 (t, J=6 Hz, 3H), 3.40-3.48 (m, 1H), 3.68-3.75 (m, 1H),
4.15 (q, J=7.11, 7.17 Hz, 2H), 4.62-4.70 (m, 1H), 7.48-7.57 (m,
5H), 7.66-7.69 (m, 1H), 7.84-7.93 (m, 4H), 9.02 (d, J=7.8 Hz, 1H);
.sup.13C NMR (75.5 MHz, CDCl.sub.3): .delta. 14.4, 29.9, 52.6,
61.4, 127.2, 127.7, 128.7, 129.5, 132.0, 133.8, 134.5, 136.4,
166.8, 170.5, 191.0; HRMS m/z (M+H).sup.+ calcd. 358.1113, found
358.1106.
Representative Procedure for Synthesis of Compound 18 (Protected
Analog 17 Coupling with Glycine)
##STR00045##
[0115] Preparation of Phenyl acetyl-5-trityl-L-cysteine: To a
suspension of S-trityl-L-csyteine 1c (4.4 g, 12 mmol) in chloroform
(92 mL) containing triethylamine (2.7 g, 26.4 mmol) cooled in ice,
was added a solution of phenylacetyl chloride (1.8 g, 12 mmol) in
chlorform (20 mL). The mixture was stirred at 0-5.degree. C. for 15
min. and at room temperature for 24 hrs. Water was added (100 mL)
and pH was adjusted to 1.5 with 5 N aqueous HCl. The aqueous phase
was removed and the organic phase was washed with saturated sodium
chloride (100 mL), dried (Na.sub.2SO.sub.4) and concentrated to
give a white crystalline solid (4.9 g) in 85% yield. Phenyl
acetyl-5-trityl-L-cysteine: m.p. 60-62.degree. C.;
[.alpha.].sub.D=+21.8.degree. (c 2, CH.sub.3OH); .sup.1H NMR (300
MHz, CDCl.sub.3): .delta. 2.60-2.71 (m, 2H), 3.5 (s, 1 H),
4.15-4.23 (m, 1H), 5.92 (d, J=6.48 Hz, 1H), 7.21-7.33 (m, 20H); );
.sup.13C NMR (75.5 MHz, CDCl.sub.3): .delta. 32.9, 43.1, 51.4,
67.8, 126.8, 127.2, 127.4, 127.8, 127.9, 128.4, 128.9, 129.1,
129.4, 144.1, 171.5, 172.5.
[0116] N-Carbobenzoxy-5-trityl-L-cysteinylglycine ethyl ester (17):
To a solution of glycine ethyl ester hydrochloride (1.25 g, 9 mmol)
in chloroform (50 mL) and triethylamine (1.25 mL) was added phenyl
acetyl-5-trityl-L-cysteine (4.8 g, 10 mmol) and
N,N'-dicyclohexycarbodiimide (2.1 g, 10 mmol). After stirred at
room temperature overnight followed by addition of a few drops of
50% acetic acid the insoluble precipitate of dicyclohexylurea (1.7
g) was removed by filtration; the filtrate was washed successively
with dilute hydrochloric acid, potassium hydrogen carbonate and
water, dried over sodium sulfate and evaporated to dryness. The
residue was treated with ethyl acetate. Some undissolved material
(dicyclohexylurea, 0.5 g) was filtered off and the filtrate was
concentrated in vacuo to a small volume. Crystalline 17 was
separated out in 85% yield. 17: m.p. 152.degree. C.; .sup.1H NMR
(300 MHz, CDCl.sub.3): .delta. 1.23-1.32 (m, 3H), 2.57-2.62 (m,
2H), 3.53 (s, 1H), 3.87-3.91 (m, 2H), 4.13 (d, J=6.18 Hz, 1H),
4.15-4.23 (m, 2H), 5.91 (d, J=7.41 Hz, 1H), 6.55 (s, 1H), 7.21-7.45
(m, 20H); .sup.13C NMR (75.5 MHz, CDCl.sub.3): .delta. 14.0, 33.0,
41.3, 43.3, 51.9, 61.4, 67.0, 126.8, 127.3, 127.9, 128.9, 129.3,
129.5, 134.1, 144.3, 169.1, 169.9, 171.1.
[0117] Bis[(R)-ethyl
2-(3-mercapto-2-(2-phenylacetamido)propanamido)acetate] (18): was
prepared in 72% yield following the procedure for preparation of 7a
and obtained as a yellow solid. 18: m.p. 98.degree. C.; .sup.1H NMR
(300 MHz, CDCl.sub.3); 81.27-1.31 (m, 1H), 2.79-2.87 (m, 1H),
3.00-3.07 (m, 1H), 3.64 (s, 2H), 3.68-3.76 (m, 1H), 3.96-4.16 (m,
1H), 4.04-4.23 (m, 2H), 5.52-5.58 (m, 1 H), 6.56 (d, J=9.15 Hz,
1H), 7.25-7.35 (m, 5H), 8.40-8.44 (s, 1H); .sup.13C NMR (75.5 MHz,
CDCl.sub.3): .delta. 14.1, 41.1, 43.1, 46.3, 53.0, 61.2, 127.2,
128.6, 129.5, 134.2, 169.1, 170.5, 171.5.
Example 5
[0118] PCP dose-dependently alters prepulse inhibition and impact
of N-acetyl cysteine on sensorimotor gating deficits produced by
PCP. Sensorimotor gating, a process compromised in schizophrenic
patients, is often measured using prepulse inhibition whereby a
mild auditory stimulus (prepulse, 2-15 db above background)
precedes (100 ms) a startle-eliciting auditory stimulus (50 dB
above background). Intact sensorimotor gating will result in
suppression of the startle reflex when preceded by the prepulse.
Since improvement in prepulse inhibition tracks improvement in
symptoms that are largely insensitive to current treatments, this
paradigm has become one of the most commonly used screening
paradigms. FIG. 4 illustrates the capacity of PCP to disrupt
prepulse inhibition, rendering the prepulse ineffective in
suppressing the startle reflex. PCP is commonly used to disrupt
prepulse inhibition because this abnormality, in addition to
negative and cognitive symptoms, are insensitive to 1.sup.st
generation antipsychotics thereby providing predictive
validity.
[0119] FIG. 5 illustrates the impact of N-acetyl cysteine on
sensorimotor gating deficits produced by phencyclidine administered
orally (left) or directly into the prefrontal cortex (right), which
is likely the therapeutic site of action for cysteine prodrugs.
N=6-46/group. * indicate a significant difference from rats
receiving PCP only (e.g., 0 N-acetyl cysteine), Fisher LSD, p,
05.
Example 6
[0120] Efficacy of compounds from scheme 1 relative to N-acetyl
cysteine in reversing PCP-induced deficits in sensorimotor gating
in rats. FIG. 6 is a bar graph illustrating inhibition of a startle
response in response to a load stimulus (pulse) when preceded by a
pre-pulse stimulus (2-15 db above background). Prepulse inhibition
is a commonly used paradigm to screen antipsychotic agents for use
in treating schizophrenia. The pre-pulse stimulus presented at 15
dB above background reduced the startle response in saline controls
(S; N=46) by >60% relative to the response elicited following
exposure to the pulse only. Rats pretreated with phencyclidine only
(P; 1, 25 mg/kg, SC; N=42) failed to exhibit a reduction in the
response elicited by the pulse even when preceded by the pre-pulse
(regardless of stimulus intensity). This reflects sensorimotor
gating deficits common to patients afflicted with schizophrenia.
Rats pretreated (60 min) with N-acetyl cysteine (30 mg/kg, po)
failed to exhibit sensorimotor gating. Note direct delivery of
N-acetyl cysteine into the brain reverses phencyclidine-induced
deficits in sensorimotor gating, which is consistent with clinical
trials establishing the antipsychotic efficacy of this compound.
Rats pretreated (60 min) with compounds synthesized from scheme 1
(N=7-22/group), notably compounds 5a-D and 4a, exhibited a
significant difference relative to either rats receiving PCP alone
(*, Fisher LSD, p<0.05) and/or N-acetylcysteine (N 30; 30 mg/kg;
+, Fisher LSD, p<0.05). Collectively, these data indicate the
efficacy of these compounds and this synthesis scheme to generate
novel antipsychotics that exceeds the potential of N-acetyl
cysteine.
Example 7
[0121] Efficacy of compounds from scheme 2 relative to N-acetyl
cysteine in reversing PCP-induced deficits in sensorimotor gating
in rats. FIG. 7 is a bar graph illustrating inhibition of a startle
response in response to a load stimulus (pulse) when preceded by a
pre-pulse stimulus (2-15 db above background). Prepulse inhibition
is a commonly used paradigm to screen antipsychotic agents for use
in treating schizophrenia. The pre-pulse stimulus presented at 15
dB above background reduced the startle response in saline controls
(S; N=46) by >60% relative to the response elicited following
exposure to the pulse only. Rats pretreated with phencyclidine only
(P; 1, 25 mg/kg, SC; N=42) failed to exhibit a reduction in the
response elicited by the pulse even when preceded by the pre-pulse
(regardless of stimulus intensity). This reflects sensorimotor
gating deficits common to patients afflicted with schizophrenia.
Rats pretreated (60 min) with N-acetyl cysteine (30 mg/kg, po)
failed to exhibit sensorimotor gating. Note direct delivery of
N-acetyl cysteine into the brain reverses phencyclidine-induced
deficits in sensorimotor gating, which is consistent with clinical
trials establishing the antipsychotic efficacy of this compound.
Rats pretreated (60 min) with compounds synthesized from scheme 2
(N=7-14/group), notably compounds 5a and 7a, exhibited a
significant difference relative to either rats receiving PCP alone
(*, Fisher LSD, p<0.05) and/or N-acetylcysteine (N 30; 30 mg/kg;
+, Fisher LSD, p<0.05). Collectively, these data indicate the
efficacy of these compounds and this synthesis scheme to generate
novel antipsychotics that exceeds the potential of N-acetyl
cysteine.
Example 8
[0122] Efficacy of compounds from scheme 3 relative to N-acetyl
cysteine in reversing PCP-induced deficits in sensorimotor gating
in rats. FIG. 8 is a bar graph illustrating inhibition of a startle
response in response to a load stimulus (pulse) when preceded by a
pre-pulse stimulus (2-15 db above background). Prepulse inhibition
is a commonly used paradigm to screen antipsychotic agents for use
in treating schizophrenia. The pre-pulse stimulus presented at 15
dB above background reduced the startle response in saline controls
(S; N=46) by >60% relative to the response elicited following
exposure to the pulse only. Rats pretreated with phencyclidine only
(P; 1, 25 mg/kg, SC; N=42) failed to exhibit a reduction in the
response elicited by the pulse even when preceded by the pre-pulse
(regardless of stimulus intensity). This reflects sensorimotor
gating deficits common to patients afflicted with schizophrenia.
Rats pretreated (60 min) with N-acetyl cysteine (30 mg/kg, po)
failed to exhibit sensorimotor gating. Note direct delivery of
N-acetyl cysteine into the brain reverses phencyclidine-induced
deficits in sensorimotor gating, which is consistent with clinical
trials establishing the antipsychotic efficacy of this compound.
Rats pretreated (60 min) with compounds synthesized from scheme 3
(N=7/group), namely compounds 11-a-b and 11-a-d, exhibited a
significant difference relative to either rats receiving PCP alone
(*, Fisher LSD, p<0.05) and/or N-acetylcysteine (N 30; 30 mg/kg;
+, Fisher LSD, p<0.05). Collectively, these data indicate the
efficacy of these compounds and this synthesis scheme to generate
novel antipsychotics that exceeds the potential of N-acetyl
cysteine.
Example 9
[0123] Efficacy of compounds from scheme 4 relative to N-acetyl
cysteine in reversing PCP-induced deficits in sensorimotor gating
in rats. FIG. 9 is a bar graph illustrating inhibition of a startle
response in response to a load stimulus (pulse) when preceded by a
pre-pulse stimulus (2-15 db above background). Prepulse inhibition
is a commonly used paradigm to screen antipsychotic agents for use
in treating schizophrenia. The pre-pulse stimulus presented at 15
dB above background reduced the startle response in saline controls
(S; N=46) by >60% relative to the response elicited following
exposure to the pulse only. Rats pretreated with phencyclidine only
(P; 1, 25 mg/kg, SC; N=42) failed to exhibit a reduction in the
response elicited by the pulse even when preceded by the pre-pulse
(regardless of stimulus intensity). This reflects sensorimotor
gating deficits common to patients afflicted with schizophrenia.
Rats pretreated (60 min) with N-acetyl cysteine (30 mg/kg, po)
failed to exhibit sensorimotor gating. Note direct delivery of
N-acetyl cysteine into the brain reverses phencyclidine-induced
deficits in sensorimotor gating, which is consistent with clinical
trials establishing the antipsychotic efficacy of this compound.
Rats pretreated (60 min) with compounds synthesized from scheme 4
(N=7/group), namely the intermediate to compound 14a (Inter-14a)
and compound 15f, exhibited a significant difference relative to
either rats receiving PCP alone (*, Fisher LSD, p<0.05) and/or
N-acetylcysteine (N 30; mg/kg; +, Fisher LSD, p<0.05).
Collectively, these data indicate the efficacy of these compounds
and this synthesis scheme to generate novel antipsychotics that
exceeds the potential of N-acetyl cysteine.
Example 10
[0124] Efficacy of compound from scheme 5 relative to N-acetyl
cysteine in reversing PCP-induced deficits in sensorimotor gating
in rats. FIG. 10 is a bar graph illustrating inhibition of a
startle response in response to a load stimulus (pulse) when
preceded by a pre-pulse stimulus (2-15 db above background).
Prepulse inhibition is a commonly used paradigm to screen
antipsychotic agents for use in treating schizophrenia. The
pre-pulse stimulus presented at 15 dB above background reduced the
startle response in saline controls (S; N=46) by >60% relative
to the response elicited following exposure to the pulse only. Rats
pretreated with phencyclidine only (P; 1, 25 mg/kg, SC; N=42)
failed to exhibit a reduction in the response elicited by the pulse
even when preceded by the pre-pulse (regardless of stimulus
intensity). This reflects sensorimotor gating deficits common to
patients afflicted with schizophrenia. Rats pretreated (60 min)
with N-acetyl cysteine (30 mg/kg, po) failed to exhibit
sensorimotor gating. Note direct delivery of N-acetyl cysteine into
the brain reverses phencyclidine-induced deficits in sensorimotor
gating, which is consistent with clinical trials establishing the
antipsychotic efficacy of this compound. Rats pretreated (60 min)
with a compound (18e) synthesized from scheme 5 (N=7) exhibited a
significant difference relative to either rats receiving
N-acetylcysteine (N 30; 30 mg/kg; +, Fisher LSD, p<0.05).
Collectively, these data indicate the efficacy of this compound and
synthesis scheme to generate novel antipsychotics that exceeds the
potential of N-acetyl cysteine.
Example 11
[0125] Efficacy of compound 5a-d (Scheme 1) as novel anticraving
agent. In addition to normalizing the function of the prefrontal
cortex, as demonstrated by the impact of the prodrugs on
pcp-induced sensorimotor gating deficits, the anticraving potential
of a drug can be demonstrated using the extinction/reinstatement
paradigm. In the present experiments, rats were implanted with
indwelling jugular catheters with an external port affixed slightly
posterior to the rat's shoulder blades. Tubing is used to connect a
syringe of cocaine to the external port of the indwelling catheter.
Rats are then placed into standard operant chambers (Med
Associates) and permitted to press a lever for an infusion of
cocaine (0.5 mg/kg/200 microL, IV). Once behavior is stable, rats
are permitted at least eleven 2-hr sessions to self-administer
cocaine. Afterwards, the cocaine solution is replaced with saline
in order to extinguish lever pressing. Once responding decreases to
10 or fewer lever presses/2 hr sessions for 3 out of 4 daily
sessions, rats are tested for reinstatement (relapse). To do this,
rats are placed into the operant chamber and vehicle or a
cysteine/cystine prodrug (1-60 mg/kg, p.o.; N=2-17) is
administered. Afterwards, rats then receive an injection of cocaine
(10 mg/kg, IP). Responding is then assessed for 120 min. Data
depicted in FIG. 11 illustrate that N-acetyl cysteine (IP) is
effective in producing a significant reduction in cocaine-induced
reinstatement at the doses of 30 and 60 mg/kg (IP; * indicates a
significant decrease in responding relative to rats treated with 0
NAC, Fisher LSD). FIG. 12 demonstrates that N-acetylcysteine is
less effective when given orally. Further, administration of 1
mg/kg of Compound 5a-d (Scheme 1) was sufficient to block
cocaine-induced reinstatement, an effect that was comparable to 30
mg/kg NAC (* indicates a significant decrease in responding
relative to rats treated with 0 NAC, Fisher LSD).
[0126] While this invention has been described in conjunction with
the various exemplary embodiments outlined above, various
alternatives, modifications, variations, improvements and/or
substantial equivalents, whether known or that are or may be
presently unforeseen, may become apparent to those having at least
ordinary skill in the art. Accordingly, the exemplary embodiments
according to this invention, as set forth above, are intended to be
illustrative, not limiting. Various changes may be made without
departing from the spirit and scope of the invention. All
publications, patents and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *